Recombinant Hordeum vulgare Low molecular mass early light-inducible protein HV90, chloroplastic

What is the structure and amino acid composition of recombinant ELIP HV90?

Recombinant Hordeum vulgare low molecular mass early light-inducible protein HV90 is a chloroplastic protein with a specific amino acid sequence: VRAQTEGPSAPPPNKPKASTSIWDEMAFSGPAPERINGRLAMVGFVTALAVEAGRGDGLL SQLGSGTTGQAWFAYTVAVLSMASLVPLLQGESAEGRAGIMNANAELWNGRFAMLGLVALAATEIITGAPFINV . The mature protein spans amino acids 39-172 of the complete sequence, with the N-terminal portion likely serving as a chloroplast transit peptide . The protein contains transmembrane domains with light-harvesting complex (LHC) motifs that are essential for its function .

What environmental factors regulate ELIP HV90 expression in barley?

ELIP HV90 expression is primarily regulated by several key environmental factors:

• Light intensity: ELIP mRNA levels directly correlate with sunlight intensity prior to sample collection, with higher expression on bright days .

• Light quality: While high light triggers expression, studies on ELIP homologs show that UV-A irradiation significantly increases ELIP protein abundance (within 1 hour at 23°C and after 3 hours at 4°C), whereas UV-B irradiation does not induce accumulation due to rapid cell death .

• Temperature: Cold stress (4°C) enhances ELIP expression and protein accumulation. Studies have shown that ELIP mRNA is detected at 3 hours after incubation at 4°C and gradually increases over time, while protein accumulation is observed from 6 hours in light and from 9 hours in dark conditions .

• Combined stresses: The combined effect of high light and low temperature results in significantly higher ELIP expression than either stress alone, indicating a synergistic effect .

• Hormonal regulation: Methyl jasmonate (JA-Me) treatment reduces ELIP transcript levels to approximately 25% after 1 day of incubation and further decreases to 5-8% by day 3, demonstrating strong hormonal repression .

How does ELIP HV90 expression change during leaf development and senescence?

ELIP HV90 expression remains responsive to light stress regardless of the developmental stage of barley leaves. Comparative analysis of flag leaves at different developmental stages in both spring and winter barley varieties demonstrates that light-stress-regulated ELIP gene expression is independent of leaf developmental stage .

Remarkably, even during senescence—when chlorophyll content, photosystem II efficiency, and 32-kD herbicide-binding protein levels decrease drastically—ELIP mRNA and protein still accumulate to high levels on bright days . This suggests that the photoprotective function of ELIP remains important throughout the entire leaf lifespan, including during the senescence phase when photosynthetic machinery is being dismantled.

What are the optimal methods for analyzing ELIP HV90 protein levels in plant samples?

For optimal analysis of ELIP HV90 protein levels in plant samples, researchers should employ the following methodological approach:

• Protein extraction and quantification:

  • Use appropriate buffers containing protease inhibitors to preserve protein integrity during extraction from leaf tissue

  • Quantify total protein content using standard methods (Bradford assay or BCA)

• Western blot analysis (as used in studies with ELIP homologs):

  • Separate proteins by SDS-PAGE

  • Transfer to appropriate membranes (PVDF or nitrocellulose)

  • Use specific antibodies raised against ELIP HV90

  • Employ ATPβ as a loading control to normalize results

  • Perform densitometric analysis for quantitative comparison

• Time point considerations:

  • Account for the time delay between mRNA expression and protein accumulation (approximately 3-6 hours)

  • When analyzing responses to environmental stresses, collect samples at multiple time points (studies show expression of ELIP mRNA is detected at 3h after cold stress, while protein accumulation occurs from 6h in light and 9h in dark conditions)

• Statistical analysis:

  • Use two-way ANOVA with Bonferroni's post hoc test for multiple comparisons

  • For comparing two groups, use two-tailed unpaired Student's t-test with Welch's correction

  • Accept statistical significance when P < 0.05

How can researchers effectively measure photoinhibition in studies involving ELIP HV90?

To effectively measure photoinhibition in studies involving ELIP HV90, researchers should utilize the following parameters and techniques:

• Photochemical efficiency measurements:

  • Measure the maximum photochemical efficiency of PSII in dark-adapted state (Fv/Fm)

  • Track changes in Fv/Fm during high light exposure and recovery periods

  • Record that in control studies, Fv/Fm decreases by approximately 55% during 4h of high light (2,500 μmol m^-2 s^-1) and recovers to 85% of initial value during 6h of recovery under low light (100 μmol m^-2 s^-1)

• D1 protein degradation analysis:

  • Monitor D1 protein levels via western blotting as a direct indicator of PSII damage

  • Compare D1 degradation rates between control and ELIP-modified samples

  • Note that D1 degradation correlates with decreased photochemical efficiency

• Rapid Light Curves (RLC) protocol:

  • Construct RLCs based on nine increasing actinic light levels (0, 16, 64, 128, 192, 320, 512, 832, 1088, 1344 μmol photons m^-2 s^-1)

  • Calculate effective quantum yield (ΦPSII = ΔF/Fm′ = (Fm′- F)/Fm′)

  • Calculate maximum quantum yield (Fv/Fm = (Fm - F0)/Fm)

  • Determine relative electron transport rate (rETR = 0.84 × 0.5 × ΦPSII × light intensity)

• Non-photochemical quenching (NPQ) assessment:

  • Calculate NPQ as (Fm′max - Fm′)/Fm′ to account for higher Fm′ values than Fm values measured after dark-adaptation

  • Compare NPQ values between control and experimental samples as an indicator of energy dissipation capacity

How does ELIP HV90 interact with the photosynthetic apparatus under stress conditions?

ELIP HV90 interacts with the photosynthetic apparatus under stress conditions through several proposed mechanisms:

• Pigment binding and protection:

  • ELIPs have been shown to bind chlorophyll a and lutein

  • They function as transient pigment carriers or chlorophyll exchange proteins during photosystem repair and reorganization

  • This binding capability helps prevent the formation of free chlorophyll molecules that could generate reactive oxygen species

• Association with photosystem components:

  • ELIPs associate with the monomeric and trimeric major LHCb antenna of PSII

  • They provide protective functions for the chloroplast during photooxidative stress under high light conditions

  • Their presence correlates with increased survival rates under combined high light and low temperature stress

• Xanthophyll cycle modulation:

  • Evidence suggests ELIPs may modulate the xanthophyll cycle, which is critical for non-photochemical quenching

  • Under stress conditions, increases in β-carotene and zeaxanthin synthesis are observed alongside ELIP accumulation

  • This relationship suggests a role in regulating photoprotective pigment composition

• Redox state regulation:

  • Studies indicate ELIP involvement in regulating cellular redox state

  • The addition of ROS quenchers (like TEMPOL) reduces ELIP expression, suggesting redox-dependent regulation

  • This regulatory role helps protect the photosystem under photooxidative stress, particularly at low temperatures

What are the comparative characteristics of ELIP HV90 and its homologs across different plant species?

The comparative analysis of ELIP HV90 and its homologs across different plant species reveals important evolutionary and functional insights:

Key comparative findings:

• Structural conservation: All ELIP homologs contain chlorophyll-binding motifs, but with variations in transmembrane domains and binding affinities .

• Regulatory differences: While all ELIPs respond to light stress, their specific induction factors vary. Some are strictly cryptochromes-dependent, while others respond to diverse signals .

• Evolutionary adaptations: Species-specific adaptations are evident in the response thresholds and expression patterns, reflecting evolutionary adaptation to different ecological niches.

• Functional conservation: Despite differences, the core photoprotective function appears conserved across species, suggesting fundamental importance in photosynthetic organisms .

What methodological challenges exist in studying structure-function relationships of ELIP HV90?

Several significant methodological challenges exist in studying the structure-function relationships of ELIP HV90:

• High sequence similarity among resistance genes:

  • More than 80% similarity in the sequences of resistance genes considerably hampers sequencing efforts

  • This high homology makes it difficult to design specific primers for targeted gene analysis

  • Researchers have reported difficulties in obtaining complete sequences of disease resistance genes due to this issue

• Transient expression patterns:

  • The transient nature of ELIP expression under stress conditions makes timing of experiments critical

  • Protein levels fluctuate based on environmental conditions, complicating consistent isolation

  • A time delay exists between mRNA expression and protein accumulation (approximately 3 hours for mRNA detection after cold stress versus 6-9 hours for protein accumulation)

• Membrane protein purification challenges:

  • As a chloroplastic membrane protein, ELIP HV90 presents inherent difficulties for structural studies

  • Traditional crystallization and structural analysis techniques may be inadequate

  • Maintaining protein integrity during extraction from thylakoid membranes requires specialized approaches

• Functional redundancy:

  • The presence of both low and high molecular mass ELIP families with potentially overlapping functions complicates loss-of-function studies

  • Determining specific functions of each ELIP type requires sophisticated genetic approaches

  • Complete analysis may require simultaneous modification of multiple ELIP genes

• Environmental variability in field studies:

  • Field studies show that ELIP mRNA levels relate to sunlight intensity before sample collection, introducing variability

  • Temperature effects can mask light responses, as protein levels do not always correlate with mRNA on days with high temperatures

  • These environmental interactions necessitate controlled growth chamber studies to complement field observations

How might ELIP HV90 research inform bioengineering strategies for enhancing crop stress tolerance?

ELIP HV90 research provides several promising avenues for bioengineering enhanced stress tolerance in crops:

• Targeted overexpression strategies:

  • Studies with ELIP homologs show that overexpression mutants survive significantly longer under combined high light and cold stress conditions

  • Modulating ELIP expression in crops could enhance photoprotection during environmental extremes

  • Tissue-specific or stress-induced promoters could optimize ELIP expression timing and localization

• Photosynthetic efficiency improvement:

  • Understanding how ELIPs protect photosystems could lead to crops with improved photosynthetic efficiency under stress

  • Research shows ELIP knockdown mutants exhibit much lower photosynthetic efficiency than wild type in low temperatures

  • Engineering optimal ELIP levels might balance photoprotection with photosynthetic capacity

• CO₂ response enhancement:

  • Research demonstrates that CO₂ influx to photobioreactors induces strong accumulation of ELIP homologs and enhances survival under high light and cold stress

  • This finding suggests potential synergies between elevated CO₂ and stress protection mechanisms

  • Crops could be engineered to better couple carbon fixation with photoprotective responses

• Multi-stress tolerance development:

  • ELIP's role in both light and temperature stress responses makes it a valuable target for multi-stress tolerance

  • Engineering crops with optimized ELIP regulation could provide protection against combined stresses that increasingly occur with climate change

  • The developmental stage independence of ELIP expression suggests these improvements could benefit crops throughout their lifecycle

• Redox homeostasis improvement:

  • ELIP involvement in cellular redox state regulation offers opportunities to enhance ROS management in crops

  • Engineered crops with improved redox homeostasis would better withstand multiple abiotic stresses

  • This approach could be particularly valuable for crops grown in marginal lands or extreme environments

How do methyl jasmonate treatments affect ELIP expression and photosystem protection?

Methyl jasmonate (JA-Me) treatments have complex and sometimes counterintuitive effects on ELIP expression and photosystem protection:

• Differential effects on ELIP expression:

  • JA-Me significantly reduces the transcript levels of both small and large ELIPs to approximately 25% of control levels after 1 day of treatment

  • This repression continues with ELIP mRNA declining further to 5-8% of control levels by day 3

  • The repression occurs despite increased photoinhibition, which normally enhances ELIP expression

• Paradoxical impact on light stress:

  • JA-Me treatment induces symptoms similar to norflurazon bleaching, including pigment loss and enhanced light stress

  • These conditions would typically increase ELIP expression, yet JA-Me simultaneously represses ELIP transcription

  • This creates a situation where photosystems experience greater stress but have reduced photoprotective ELIP expression

• Photosystem II efficiency impairment:

  • In JA-Me-treated leaf segments, the decrease in photochemical efficiency (Fv/Fm) under high light is substantially more pronounced compared to controls

  • After 4 hours of high light exposure, Fv/Fm in JA-Me-treated tissues approaches zero, while controls maintain higher values

  • Recovery of photosynthesis occurs substantially more slowly in JA-Me-treated tissues, reaching only about 65% of control values

• D1 protein dynamics:

  • JA-Me treatment results in massive degradation of D1 protein under high light conditions

  • Recovery of photosystem II activity is significantly delayed compared to controls

  • This suggests JA-Me creates a condition where D1 turnover is impaired, possibly due to reduced photoprotection from ELIPs

• Hierarchical gene regulation:

  • JA-Me represses light stress-induced ELIP expression more slowly than it represses constitutively expressed genes like LHC II and SSU

  • This indicates a hierarchical regulation where stress-responsive genes maintain inducibility longer than housekeeping genes under hormone treatment

  • The delayed repression of ELIPs suggests their critical importance in plant stress responses

What genetic mapping and isolation techniques have been applied to study ELIP-related resistance genes in barley?

Advanced genetic mapping and isolation techniques have been applied to study ELIP-related resistance genes in barley, particularly for leaf rust resistance genes:

• High-resolution mapping populations (HRMP):

  • Construction of segregating populations comprising 4,775 F₂ plants for fine mapping

  • Development of 537 recombinant inbred lines (RILs) achieving genetic resolution of 0.010% recombination

  • Phenotypic analysis of resistance showing 1:1 segregation ratio (261 resistant and 276 susceptible RILs)

• Marker saturation technologies:

  • Initial use of Simple Sequence Repeats (SSRs), size polymorphism markers, and SNPs from barley GZ and 9K iSelect chips

  • Advanced saturation using Illumina 50K Infinium array and Genotyping By Sequencing (GBS)

  • Integration with barley reference sequence for precise marker placement

• Kompetitive Allele Specific PCR (KASP) assay development:

  • Conversion of polymorphic SNPs to KASP assays

  • Design of allele-specific forward primers and common reverse primers spanning SNP positions

  • Application of Primer3 v.0.4.0 for optimal primer design

• High-throughput genotyping:

  • Screening with 50K chip revealing 40,777 scoreable SNPs across the barley genome

  • Identification of 14,616 homozygous polymorphic SNPs between resistant and susceptible genotypes

  • Detection of 39 SNPs in the large interval and 4 SNPs in the closest target interval

• Candidate gene identification and validation:

  • Narrowing of target interval to 0.44 Mb containing 11 low confidence and 18 high confidence genes

  • Functional annotation revealing 5 genes related to pathogen resistance

  • Allele-specific re-sequencing identifying 259 SNPs in disease resistance genes, including functionally significant mutations (11 synonymous mutations, 17 amino acid substitutions, and 2 arginine-to-stop codon changes)

How do ELIP expression patterns in controlled laboratory conditions compare with field observations?

Comparative analysis reveals significant differences and similarities between ELIP expression patterns in controlled laboratory conditions versus field observations:

Key insights from this comparison:

• Validation requirement: Laboratory findings require field validation to confirm ecological relevance

• Complementary approaches: Both approaches provide unique insights that together create a comprehensive understanding

• Temporal considerations: Field studies reveal seasonal patterns not captured in short-term laboratory experiments

• Stress complexity: Field conditions present more complex combinations of stresses that may reveal interactions not obvious in controlled settings

• Practical applications: Field observations are essential for translating laboratory findings into agricultural applications

What functional differences exist between native and recombinant forms of ELIP HV90?

Understanding the functional differences between native and recombinant forms of ELIP HV90 is crucial for interpreting research findings:

• Structural considerations:

  • Recombinant ELIP HV90 typically includes a His-tag or other fusion tags that may affect protein folding or interactions

  • Commercial recombinant proteins are expressed in E. coli systems rather than plant cells, potentially affecting post-translational modifications

  • The recombinant form often represents just the mature protein (amino acids 39-172) without the transit peptide found in the native pre-protein

• Localization differences:

  • Native ELIP HV90 is synthesized with a chloroplast transit peptide and undergoes targeting and processing

  • The native protein is integrated into thylakoid membranes within chloroplasts

  • Recombinant proteins lack the cellular context for proper membrane integration

• Post-translational modifications:

  • Native ELIPs in plants undergo phosphorylation and other modifications in response to light conditions

  • Recombinant proteins expressed in bacterial systems lack these plant-specific modifications

  • These differences may affect protein stability, turnover, and interaction capabilities

• Pigment binding capabilities:

  • Native ELIPs bind chlorophyll a and lutein within the chloroplast environment

  • Recombinant proteins expressed in E. coli lack access to these pigments during expression

  • In vitro reconstitution with pigments is possible but may not fully replicate the native configuration

• Experimental applications:

  • Recombinant proteins are valuable for in vitro binding studies, antibody production, and structural analysis

  • Native proteins in their cellular context are essential for understanding physiological function and regulation

  • Complementary approaches using both forms provide the most complete understanding of ELIP HV90 function